Cargando…
A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway
DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials ar...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582407/ https://www.ncbi.nlm.nih.gov/pubmed/30869144 http://dx.doi.org/10.1093/nar/gkz171 |
_version_ | 1783428312721260544 |
---|---|
author | Healing, Eleanor Charlier, Clara F Meira, Lisiane B Elliott, Ruan M |
author_facet | Healing, Eleanor Charlier, Clara F Meira, Lisiane B Elliott, Ruan M |
author_sort | Healing, Eleanor |
collection | PubMed |
description | DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactive and quantitative determination of repair enzyme activity at individual steps and over multiple steps of the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrations of 0.0002 to 0.181 μg per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk. |
format | Online Article Text |
id | pubmed-6582407 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-65824072019-06-21 A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway Healing, Eleanor Charlier, Clara F Meira, Lisiane B Elliott, Ruan M Nucleic Acids Res Methods Online DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactive and quantitative determination of repair enzyme activity at individual steps and over multiple steps of the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrations of 0.0002 to 0.181 μg per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk. Oxford University Press 2019-06-20 2019-03-14 /pmc/articles/PMC6582407/ /pubmed/30869144 http://dx.doi.org/10.1093/nar/gkz171 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Healing, Eleanor Charlier, Clara F Meira, Lisiane B Elliott, Ruan M A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway |
title | A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway |
title_full | A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway |
title_fullStr | A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway |
title_full_unstemmed | A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway |
title_short | A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway |
title_sort | panel of colorimetric assays to measure enzymatic activity in the base excision dna repair pathway |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582407/ https://www.ncbi.nlm.nih.gov/pubmed/30869144 http://dx.doi.org/10.1093/nar/gkz171 |
work_keys_str_mv | AT healingeleanor apanelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway AT charlierclaraf apanelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway AT meiralisianeb apanelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway AT elliottruanm apanelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway AT healingeleanor panelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway AT charlierclaraf panelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway AT meiralisianeb panelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway AT elliottruanm panelofcolorimetricassaystomeasureenzymaticactivityinthebaseexcisiondnarepairpathway |